The major long range purpose of the studies initiated in this R21 proposal is to characterize the erythroid alternative splicing program, including (a) identification of erythroid stage-specific switches in alternative pre- mRNA splicing, and (b) elucidating the role of major splicing regulatory proteins in effecting this program. The alternative splicing program is hypothesized to play a major role in regulating structure of key erythroid proteins, and these studies may therefore stimulate functional studies of novel erythroid protein isoforms. Proof of principle for these concepts is provided by previous studies of a stage-specific switch in alternative pre-mRNA splicing in the protein 4.1R gene that is critical for erythroid membrane mechanical properties. Previous genome-wide analyses of erythroid gene expression have provided valuable quantitative data on the expression levels of each gene. However, existing technologies for quantitating mRNA output from each gene fail to distinguish qualitative differences in mRNA, generated by alternative splicing, which may completely alter gene function. In this R21 application, novel Affymetrix microarrays with the capacity to interrogate virtually all known and predicted exons will be used to study programmed changes in alternative splicing during erythropoiesis. Preliminary collaborative experiments using the prototype research version of the arrays confirm that they efficiently identify splicing differences between differentiated cell types. By extension, they should also detect stage-specific changes in splicing during differentiation of a single lineage; e.g., facilitating examination of the erythroid transcriptome with unprecedented molecular resolution. Probes prepared from cultured human and mouse erythroid progenitor RNAs at different stages of differentiation will be hybridized to the microarray and analyzed to elucidate changes in exon expression patterns. Two aims are proposed: (1) Perform a comprehensive analysis of stage-specific switches in pre-mRNA splicing that constitute the normal erythroid alternative splicing program in human and mouse models, and (2) Functionally assess the role of selected splicing factor proteins in erythroid differentiation by measuring splicing changes that occur in response to RNAi-mediated knockdown of splicing factor expression levels. Successful accomplishment of these goals should provide significant new insights into the scope of the erythroid alternative splicing program; facilitate future mechanistic studies of splicing switch mechanisms; and stimulate functional analyses of novel isoforms of erythroid cytoplasmic, cytoskeletal and membrane proteins. Genetic disturbances in splicing regulation have been described in other tissues including muscle and in brain; the present studies may provide a foundation for discovery of novel splicing defects in erythroid cells. Ultimately, methods for therapeutic correction of splicing defects, under development in model systems, may allow correction of such defects in erythroid cells as well. [unreadable] [unreadable] [unreadable]